Enzyme of use in chitosan hydrolysis

Abstract

The present invention relates to a naturally occuring, isolated and characterized microorganism producing hydrolase enzyme, specifically, chitosanase; chitosanase isolated thereof; the nucleic acid sequence of the portion of the gene encoding this chitosanase; complete amino acid sequence of this chitosanase; hybrid plasmids containing the related gene; host microorganisms transformed with the plasmids; recombinant microorganisms overexpressing the enzyme; and to enzymatic treatment of chitosan with the aforementioned enzyme resulting in molecular weight decrease, viscosity decrease and increase of solubility of chitosan in water.

What is claimed is: 
     
       1. A chitosanase isolated from Streptomyces N174 or obtained from a microorganism of the genus Streptomyces which produces said chitosanase, the chitosanase hydrolysing specifically a substrate chitosan, said chitosanase having the following characteristics: an apparent molecular weight (M r ) of 29500 kilodaltons measured by SDS-polyacrylamide gel electrophoresis;   a pH range for activity extending from 4.0 to 6.0, with a maximum of activity at pH 5.5;   an apparent constant of affinity (k m ) for said substrate chitosan of 0.088 mg/ml and a maximal velocity of hydrolysis (V max ) of 96.6 U/mg, where in U is defined as the amount of chitosanase that liberates 1 μmol of the product of said hydrolysis, D-glucosamine equivalent, in one minute of enzymatic reaction at 37° C. in a one ml volume of reaction containing 50 mM acetate buffer pH 5.5 and 0.2% solubilized chitosan having a degree of acetylation of 21%, the reaction of said substrate with said chitosanase being allowed for 10 minutes and stopped by addition of neocuproine reagent.   
     
     
       2. A chitosanase according to claim 1, having the amino acid sequence defined in SEQ. ID. No. 3. 
     
     
       3. A method of production of chitosanase as claimed in claim 1, comprising the following steps: culturing a strain producing said chitosanase in a culture medium containing a carbon source which is able to support growth of said strain and production of chitosanase;   recovering said chitosanase secreted in said culture medium; and   purifying said chitosanase by adding a solution of polyacrylic acid to said medium which precipitates said chitosanase.   
     
     
       4. A method according to claim 3 wherein the solution of polyacrylic acid has a concentration of 2% and said solution is added till the weight of polyacrylic reaches 4 times the weight of proteins measured in said medium. 
     
     
       5. A method according to claim 3 further characterized by a further purification of said chitosanase by chromatography, said further purification achieving a substantially pure chitosanase. 
     
     
       6. A method according to any one of claims 3 to 5 wherein the strain is Streptomyces N174 having ATCC Deposit Number 55633. 
     
     
       7. A method of hydrolysis of chitosan by chitosanase which comprises the following steps: adding the chitosanase of claim 2 to a solution containing chitosan;   allowing hydrolysis to occur;   detecting said hydrolysis by measuring the average degree of polymerization of said chitosan.   
     
     
       8. A method according to claim 7 wherein said degree of polymerization is measured by HPLC. 
     
     
       9. A method according to claim 7 wherein said chitosanase and chitosan are mixed in the proportions of 0.006 to 0.2 IU/ml and 0.1 to 4 mg/ml, respectively in a 50 mM sodium acetate buffer, pH 5.5.